The AIDS syndrome is widely believed by the majority of Scientists (with a few exceptions) to be the end result of infection with Human Immuno Deficiency Virus type 1 or 2. Despite available treatment, on diagnosis of AIDS, life expectancy is short and death occurs usually within two years—death resulting from overwhelming infection and/or neoplastic diseases provoked by a profound immuno deficiency. The causative agent of this condition the HIV virus was proposed by Montagnier L and by Barre-Sinoussi F et al 1983, Science 220, 868-870 and slightly later by Galio R et al 1983, Science 220 865-867.
The accepted view of most Scientists is that this virus is necessary at least in part for production of this fatal Immuno Deficiency Syndrome. One notable exception to this belief is Deusberg P H who champions the minority view that AIDS is not caused by the Human Immuno Deficiency Virus and that it is not transmissible; Deusberg P H Proceedings National Acad Sci USA Vol 88, 1575-1579, February 1991. Regardless of the merits of this contradictory opinion and academic debate, work continues to find methods of combating the HIV virus and removing its presence from infected humans. Since the discovery of the HIV agent in the early 1980's screening of the worlds population has demonstrated that the HIV/AIDS pandemic is much worse than previously imagined. Evidence suggests that the impact of the disease will fall on disadvantaged women and children living in deprived urban areas or in sub-Saharan Africa or the Third World. It is estimated that 10 million persons will contract this condition before the year 2000 Chin J. Lancet 1990; 336: 221-224. Transmission of this virus to newborns from infected mothers remains a growing problem. At the present time in Africa, there are at least 2.5 million infected females who have given birth to an estimated 0.5 million infected infants. Disadvantaged urban areas in the developed world such as areas of New York associated with high rates of drug use have also shown high sero-positivity rates of HIV among neonates ie 2.2% compared with an overall sero-positivity of 1.25% among neonates in that city. NOVAK, Journal of America Medical Association 1989:261:1745-1750. The social and economic costs of this epidemic are likely to overburden the resources of health care providers and remains a growing area of concern. Our understanding of the timing of maternal to fetal transmission of HIV has been greatly improved by the study of Ehrnst A et al Lancet, 1981; 338: 203-207. He demonstrated effectively that the virus is transmitted vertically at the time of or close to delivery. Despite high viral titre levels during pregnancy it seems that the mixing of maternal and fetal blood at time of delivery is the most important mode of transmission from mother to fetus. In other contexts viral titre levels are directly related to disease severity; studies by Ho D D “Quantitation of Human Immuno Deficiency Virus type 1 in the blood of infected persons.” New England Journal of Medicine 1989; 1621-1625 and the study by Combes R W et al “Plasma Vireama in Human Immuno Deficiency Infections” New England Journal of Medicine 1989; 321: 1626-1631. Both these studies have demonstrated rising levels of plasma vireama throughout the course of HIV and have suggested that high viral titres are associated both with the degree of immuno suppression, the presence or absence of symptoms and the likelihood of transmission to others. It has been shown that in the early stages of infection, viral titre levels measure 50 virus particles per ml rising to 10-50,000 virus particles per ml or more at the onset of AIDS.
In addition to the offspring of infected mothers, other large groups of the population are at risk or believe themselves to be at risk from the infection, especially homosexuals, drug addicts, persons receiving blood transfusions and health care workers. In the case of health care workers, the risk from a health care procedure carried out on infected persons is likely to be very low and is presently estimated to range of 0.3%-0.5% following needlesticks with an infected needle or surgical instrument. Operating room personnel frequently exposed to needlestick and other injuries are especially concerned at the present time. 8This concern is most marked among Orthopaedic Surgeons who in addition to needlestick injuries are liable to suffer abrasions or even transfixion of a digit with steel wires, pins and the like. So great is the concern that chain-male gloves have been devised to protect staff from inadvertent needlestick injuries. Disadvantages are apparent with barrier methods in relation to HIV prevention. In particular, increased costs—it is known that the dry goods budget of many hospitals has increased considerably following the onset of this epidemic. A second disadvantage is the loss of tactile sensation, making operative procedures more difficult and time consuming. A further problem is the difficulty in attracting and keeping staff in high risk areas who report psychological stress on their part or the part of their spouses, resulting from risk either real or imagined, particularly following needlesticks. A further area of concern is the safety of blood transfusions and the blood supply both in the US and elsewhere. To prevent repeating the iatrogenic infection of large numbers such as haemophiliacs with infected pooled factor 8, there has been a growing awareness of the need to take additional precautions with whole blood and blood product transfusion. The mainstay of transfusion safety remains screening, however no amount of screening can guarantee the safety of a blood transfusion or a blood derived product. Accordingly many workers have devised different methods to treat blood for donation. One is directed to the methods of Sieber U.S. Pat. No. 4,915,683 who discloses for example photosensatising agents useful to neutralise viruses and other pathogens in blood for transfusion. This approach and other similar methods is non specific however and requires an irradiating component in addition to a chemical means of treatment.
The present disclosure provides a means of improving the safety of blood products in one step.
The mechanism of HIV infection involves the binding of viral GP120 or envelope peptide to the CD4 peptide molecule. The CD4 protein is a 433 amino acid transmembrane glycoprotein which is found predominantly of the surface of T4 helper cells. It is also found on the surface of the mega karyocytes, monocytes, macrophages and other cells.
The CD4 molecule has three parts: an extracellular part which is divided into 4 variable domains V1 being responsible for the binding of HIV GP120; a short transmembrane region and an intracellular domain.
The CD4 molecule is part of the immunoglobulin super family. That CD4 was the receptor for the virus was shown by Dalglaoise et al 1984. “The CD4 antigen is an essential component of the receptor for the AIDS retrovirus” Nature: 212: 763-766. Also Madden P J Dalglaoise A G McDougall J S et al “The T4 gene encloses the AIDS virus receptor and is expressed in the immune system and the brain” Cell: 47, 333-348.
Maddon P et al Cell: Vol 47, 333-348 have shown that transformed mouse cells expressing the CD4 molecule possess the capabilities of binding the HIV virus though lack the ability to support the HIV infection at a cytoplasmic level. Debate continues within the art as to the precise binding site of GP120 with the CD4 molecule. Arthus et al Cell: Vol 57, 469-491 May 5, 1989 proposes that the binding site is contained at least within the first 106 amino acids of the CD4 molecule ie the V1 domain. He bases his assumption on substitute mutants and binding analysis with soluble truncated proteins demonstrating a high binding affinity solely within the first 106 amino acids of the CD4 molecule. Substitution studies led to the notion that the primary binding site on the CD4 molecule lay between amino acids 41-55.
Mizukani et al Proc Nat Acad Sci USA 85; 9273 (1988) and Arthus 1989 “Identification of the residues in human CD4 critical for the binding of HIV” Cell 57; 469 believes that the folded structures of the CD4 molecule brings other amino acids into play. Areas likely to be important in vital CD4 binding are reported to lie between amino acids 41-47 amino acids 16-49, 31-63 and 74-94 of the CD4 molecule.
The remarkable specificity of the virus for the CD4 molecule has lead to the attempts by many workers to utilise CD4 based peptides as therapeutic agents. The binding affinity for the GP120 with CD4 lies in the region of 3 nM. Many efforts have been made to utilise this high specificity.
In the case of HIV 2, the affinity is 20-25 fold less, however the binding strength as described is such that even a 25 fold reduction of affinity would not reduce the usefulness of therapeutic molecules based on CD4 and for that reason the binding reaction between GP120 and CD4 remains a logical target for anti HIV pharmaceuticals.
However the nature of the molecule means that a linear peptide will be unlikely to be successful in a therapeutic context.
Brodsky et al Immunology Vol 144, 3078-3086 Apr. 15, 1990 No 8 suggests that CD4 based therapeutic molecules should contain amino acids at least from the first 117 amino acids of the CD4 sequence.
Moore J P AIDS 1990, 4: 297-3-5 believes that the CD4 GP120 binding reaction is the one step this virus cannot afford to change by mutation to any appreciable extent.
Accordingly, attempts have been made to use recombinant CD4 peptides as therapeutic agents.
Fisher R et al Nature Vol 331, 7 Jan. 1989, “HIV Infection is blocked in vitro by recombinant soluble CD4” reported the use of recombinant soluble CD4 peptides that showed viral blocking activity in vitro. Hussey R et al “A Soluble CD4 protein selectivity inhibits HIV replications and Syncitium formation” Nature 331, 7 Jan. 1989, reported encouragingly that Syncitium formation could be blocked in vitro by the same CD4 based soluble peptides; and Dean et al “Soluble CD4 protein inhibits HIV virus infection” Nature 331, 7 Jan. 1989: A Traunecker et al “Soluble CD4 molecules neutralise Human Immuno Deficiency Virus type 1”, also Nature Vol 331, 7 Jan. 1989 encouragingly reports that CD4 peptides may be used as therapeutic agents. The euphoria provided initially by these experiments was short lived.
Problems emerged, in particular the rapid clearance of recombinant soluble CD4 or sCD4 from the blood stream and secondly the existence of multiple binding sites on the virus. It is known that the virus contains no less than a 72 GP120 molecules projecting from its surface. Therefore to fully neutralise a virus particle it would be necessary to combine recombinant soluble CD4 peptides or peptide based molecules with all 72 of the GP120 molecules the kinetics of this event are unfavourable. The present disclosure provides means to overcome both of these problems.
Further discouragement was provided by Eric Daar et al. Proc Nat Acad Sci USA Vol 87, 6574-6578. “High concentrations of recombinant SCD4 are required to neutralise primary immuno deficiency type 1 isolates.” This report based on the treatment of 45 patients shows that soluble CD4 peptides failed to consistently reduce HIV titres and further suggests a reason for the failure being lower GP120-sCD4 affinities than being reported in vitro. Daar suggests that mutant strains possessing lower CD4 affinity than the strains used in laboratory testing may be responsible for the failure of the agents.
The present disclosure proposes that clinical disappointment with the T4 agents, such as disclosed in PCT WO89/019140 relates more to the short half life of the peptides in plasma and to the fact that 72 binding sites exist on a single virus, rather than due to lack of affinity between the agent and the virus itself. So great is the affinity between CD4 molecule and the GP120 molecule that even affinities 1000 fold less than those seen in vitro should be pharmaceutically effective.
Despite the failure of unmodified soluble CD4 as a HIV therapy, other workers have advanced alternatives in the hope of utilising the GP120-CD4 interaction. Examples of some of these alternatives are fusion peptides where a CD4 component is fused to another peptide having a different function.
Capon D J et al disclosed the use of CD4 fused to the FC component of the human Igh antibodies. The objective being to mobilise the compliment system and thereby destroy the virus particle by the compliment cascade. Capon refers to these new molecules as immuno adhesives. A possible disadvantage associated with this approach is the necessity of a fully functional compliment system, unlikely to the case in advanced HIV infections. See Capon et al “Designing CD4 Immunoadhesins for AIDS Therapy” Nature Vol 337.
The molecular machines of the present disclosure do not require a functional immune system and may be effective in cases where no immune function exists.
At the present time the main stay of treatment relies of the substance Azidothymine or AZT, this substance acts on the HIV enzyme reverse transcriptase.
An increasing number of patients and earlier in their illness are receiving treatment with AZT, however tolerance of this drug can be a problem. AZT is known to be lymphotoxic and is additionally associated with substantial anaemia. Haemato toxicity appears to be major limiting factor in the use of AZT in the treatment of ARC and AIDS Richman et al “The toxicity of the Azidothymine”, New England of Medicine, 317; p 192-197, 1987. So profound is the anaemia provoked by this agent that many patients require frequent blood transfusions or treatment with Epogen (R), a novel genetically engineered erythropotein. The molecular machines of the present disclosure may be used in combination with AZT or other treatments and will augment the effectiveness of blood transfusions given to patients receiving AZT who are suffering from severe anaemia. Other agents acting on the reverse transcriptase such as DDC or DDI suffer high side effect profiles also and are toxic compounds in their own right. Equally, agents acting at other sites such as glycosylation are known to exhibit severe toxic effects on the host.
The agents of the present disclosures are anticipated to have lower toxic effects on the host than existing treatment modalities.
The agents may be used alone or in combination with existing treatments.
So great is the side effect problem of anti-AIDS therapeutics many feel it is likely that no one agent will be successful in combating this disease. One is directed to Fisher World Patent WO 89/11860 for a discussion of combination therapy.
More recently Taylor R P in Proceedings National Acad Science 88, p 3305-3309 refocused our attention on the work of Nelson published in 1950's who some years ago proposed an immune function for the red cells. Red cells (erythrocytes) are often considered to have only oxygen carrying and acid base balancing functions in the blood stream. According to Nelson red cells have additional immune functions. In particular Nelson proposed that opsonised antigens ie antigens whose surfaces were bound to compliment derived peptides could be fused to the compliment 1 receptor molecule (CR1) on the red cell surface. By fusing a virus or bacterium to the red cell surface in this way the virus or antigen becomes bound to the red cell surface in the manner of a barnacle bound to the surface of a ship.
A virus immobilised in this way would be harmless and be carried around the circulation on the red cell surface to await final phagocytosis and digestion at the end of the red cells life (usually 120 days from formation). It is thought that many antigens and pathogens are cleared from circulation by this mechanism rather than by direct phagocytosis and the like so frequently discussed in immunology texts. Accordingly, Taylor R P proposed a technique to utilise this long forgotten mechanism. He suggested the use of hybrid antibodies capable of binding both to the HIV virus and to the compliment 1 receptor molecule of the red cell surface. The adherence of virus particles to the red cell surface membrane in this way would clear the blood stream of free virus particles and free GP120. Thereby attempting to achieve Nelson's proposition that one day red blood cells could be harnessed to rid mankind of infection.
The peptide machines of the present disclosure provide totally different mechanisms to achieve Nelson's dream ie to bring about the binding of HIV virus particles to the red cell surface membrane.
The agents of the present disclosure carry the additional advantage in that antibodies to the compliment peptides and red cell surface CR1 are not a feature. This would be an advantage in that the risk of destabilising an already unbalanced immune system is not expected to be a feature of the molecules of the present disclosure.
The Third World or underdeveloped world is no stranger to epidemics and has prior to the development of AIDS and HIV been ravaged by infectious disease: the most serious on a global scale is probably malaria. Various forms of importance to humans are Plasmodium Falciparum, Plasmodium Vivax, Plasmodium Ovale. Plasmodium Malariae. This serious parasite infests red blood cells. The life cycle is complex and consists in part of a short life cycle in the salivary gland of mosquitoes. Following innoculation into a human being, the malaria organism migrates to the liver and to the red blood cell. It multiplies forming further merozoites. Merozoites may then proceed to infect other red blood cells, or may find their way into another mosquito where they replicate in the mosquito salivary gland and later proceed to infect another human being.
The course of malaria is a variable one and may be characterised by a short acute illness which can bring death in a matter of hours; or a longer more chronic illness associated with debility and anaemia. Other forms of malaria infect animals such as plasmodium Knowlesi which is known to infect the Rhesus monkey; and many other zooanotic forms.
The preferred location for the malaria parasite is within the red cells of the infected host and for much of its life span it lives intracellularly protected from the host immune system.
Merozoites are thought to spend but a brief period free in the circulation. Accordingly, considerable research efforts have been expended to discover the means whereby the merozoite forms of the parasite gains attachment and gains entry into the human red cell.
Margaret E Perkins Journal Experimental Medicine 160 September 1984, 788-798 is responsible for the formulation of a relationship between the Plasmodium Falciparum glycophorin binding molecule and glycophorin A and B two silao glycoproteins found on the surface of red cells.
More laterally, Holt and Perkins et al American Journal Tropical Medicine Hygiene 1989, p 245-251 disclose species and stain variations of Plasmodium Falciparum wherein some strains of the organism exhibit preference for sialo glycoproteins glycophorins A, B and C; and also demonstrated was the varying requirements for the N-acetyl-neuramic acid residues (NeuNac).
The glycophorin A molecule is a highly glycolated peptide. Pasvol has suggested that glycophorin binding peptide binds to the region of glycophorin close to the lipid bi-layer. Pasvol G et al “Inhibition of Malaria Parasite Invasion by Monoclonical antibodies against glycophorin A correlates with a reduction in red cell membrane deformality.” Blood 74, No 5, October 1985, 1836-1843. Debate continues within the literature as to the requirement for sialic acid on the glycophorin molecule to effect invasion by merozoites.
Some strains of malaria are totally dependent on normally sialated glycophorin A to gain entry into the red cell, whereas other strains seem to be independent of sialic acid. See Mitchell et al 67, No 5 May 1986 1519-1521. Perkins and Roco in Journal of Immunology, 88, Vol 141, 3190-3196 No 9, again stress the importance of sialic acid where normally silated glycophorin is necessary to achieve successful binding of merozoite peptides, in particular to pf200.
For several years a peptide called the glycophorin binding protein was believed to be the primary peptide responsible for binding merozoites to erythrocytes. A gene coding for GBP was isolated by M Ravetch and Kochan J and disclosed in Science Vol 227, p 1593-1596.29 Mar. 1985 and incorporated herein fully by reference.
GBP 130 is characterised by a tandem repeated sequence believed to be the site of erythrocyte binding, “A tandem repeated sequence determines the binding domatin for an erythrocyte receptor binding protein of plasmodium falciparum”. Kochan J, Perkins M and Ravetch J Cell, Vol 44, 689-696 March 1986. See FIG. 2 p 691 which also discloses the full sequences and genetic code of the GBP 130 molecule and is incorporated fully herein by reference.
Other workers have challenged the supremacy of the GBP 130 as the primary binding molecule of the malaria merozoite, Orlandi P; Kim Lee Sim et al Molecular and Biochemical Parasitology, 40 (1990) 285-294 “Characterisation of the 175 kilodalton erythrocyte binding antigen of plasmodium falciparum” suggested a different peptide, the EBA 175 molecule, as being responsible for merozoite binding or at least playing some role therein. The EBA 175 molecule like the GBP 130 molecule has an affinity for the red blood cell surface and binds thereto.
It is known that the EBA 175 molecule has an affinity for oligosaccharides which are found on the surface of the red cell molecule. However a problem arises in that the EBA 175 molecule does not bind effectively with the malaria merozoite parasite. Therefore it is thought that the EBA 175 serves a function as a bridge. This disclosure proposes an alternative mechanism in that the EBA 175 molecule is responsible for bringing the merozoite close to the erythrocyte membrane surface, thereafter GBP 130 drags the merozoite closer still by binding with the base of the glycophorin A peptide; thus bringing the lipid bilayer of the malaria parasite into approximation with the lipid bilayer of the red cell membrane and thereby allowing the incorporation of the parasite into the erythrocyte itself. This disclosure suggests the merozoite is winched into the RBC cytoplasm.
The genetic sequence and the peptide sequence of EBA 175 was disclosed by Kim Lee Sim. Orlandi P et al “Primary structure of the 175 K plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion” See J Cell Biology Vol 111 (1990) p 1877-1884 FIG. 2 of P 1880 for the sequence of amino acids and DNA sequence.
To further complicate the picture Dagmar Nolte et al described two close relatives of the glycophorin binding peptide 130 molecules which they call GBPH or glycophorin binding peptide homologues. These molecules like the GBP molecule display several tandem repeat sequences and a high affinity for the erythrocyte surface membrane surface peptides. It has been proposed by Nolte and co-workers that it is the GBPH molecule and not the GBP molecule that is responsible for erythrocyte binding of the parasite in that the GBP molecule is released as an immunogenic decoy to distract the immune system from the real binding peptide the GBPH.
The nucleotide sequence and amino acid sequence of one form of the peptide GPBH is disclosed by Dagmar Nolte et al in the Journal of Molecular and Biochemical Parasitology 49, (1991) p 253-264 see FIG. 2 of p 257 incorporated herein fully by reference. See also FIG. 3 of p 258 the same journal and paper which lists a comparison between GBP 130 and GBPH. Binding and entry of merozoites into RBC's probably involves several peptides or several alternatives as fail safes for the organism.
The picture is further complicated by other research notably by Peterson Gregory who proposes PMMSA (Pre major merozoite surface antigen) as being responsible for erythrocyte binding either in this state or following fragmentation into smaller fragments. The genetic sequence and the peptide sequence of the PMMSA molecule is given in the Journal of Molecular and Biochemical Parasitology 27 (1988) 291-302 see FIG. 3 of P 294 and 295 Peterson G et al, and is incorporated fully herein by reference.
Erythrocyte binding using different peptides and surface molecules is exhibited by other species of the malaria parasite in particular the Plasmodium Vivax organism. This organism can infect only persons expressing the Duffy marker. The Duffy antigen is a red cell surface marker and is one of many blood group markers and carried by a percentage of the population. Persons not expressing Duffy antigens are therefore immune from infection by Plasmodium Vivax. The Plasmodium Vivax merozoite expresses a Duffy binding receptor molecule the P. vivax Duffy receptor, cloned and sequenced by Xiangdang Fang and disclosed in Molecular and Biochemical Parasitology 44 (1991) p 125-132 see especially FIG. 1 of p 127 for the genetic sequence and amino acid sequence. Similar to Plasmodium Vivax is Pladmodium Knowlesi which also uses the Duffy antigen. This organism parasitises Rhesus monkeys. Also in the same Journal, same figure, same page is listed the genetic sequence of Plasmodium Knowlesi Duffy receptor molecule which may find a use in the machines of the present disclosure.
When developing therapeutic agents directed against the malaria parasite itself then it is the teachings of the art to identify the precise molecule responsible for merozoite binding in the clinical context. However, this disclosure teaches that where malaria peptides are to be employed as erythrocyte binding agents more generally then it is not important to identify the precise peptide the malaria organism uses to effect invasion, rather any malaria peptide capable of binding to an erythrocyte surface membrane may have a therapeutic use for the purposes of the machines of the present disclosure and also segments of such a peptide.
It is disclosed that the machines of the present invention consists of hybrid peptides formed by the fusion of malaria derived peptide sequences sufficient to exhibit erythrocyte membrane affinity fused to a CD4 derived peptide sequence sufficient to demonstrate affinity for the HIV virus. The restriction map and peptide sequence of the CD4 molecule are disclosed by Madden et al Proceedings National Acad Science USA 84; 1987 p 1955-1959 see especially FIGS. 1 and 2 incorporated herein fully by reference.
Also Madden et al Cell Vol 42, P 93-104 August 1985 especially FIG. 6 of P 97.
It will be appreciated that the CD4 peptide components will not be restricted to segments of the naturally occurring CD4 molecule. Also, the malaria derived peptide fragments may be any that are capable of binding to the red cell membrane and one is especially directed to those referenced herein before.